Download Functional Characterization of Two Human Olfactory Receptors

Chem. Senses 30: 195–207, 2005
doi:10.1093/chemse/bji015
Functional Characterization of Two Human Olfactory Receptors Expressed
in the Baculovirus Sf9 Insect Cell System
Valéry Matarazzo1*, Olivier Clot-Faybesse1*, Brice Marcet1*, Gaëlle Guiraudie-Capraz1, Boriana
Atanasova2, Gérard Devauchelle3, Martine Cerutti3, Patrick Etiévant2 and Catherine Ronin1
1
UMR 6149 et GDR 2590 CNRS et Université de Provence, IFR du Cerveau, 31 Chemin J. Aiguier,
F-13402 Marseille Cedex 20, France, 2Laboratoire de Recherches sur les Arômes, INRA, 17 rue
Sully, BP 86510, 21065 Dijon Cedex, France and 3Station de Recherches de Pathologie
Comparée, CNRS- INRA et GDR 2590, St Christol-Lez-Alès, France
Correspondence to be sent to: Pr. Catherine Ronin, UMR 6149, CNRS, 31 Chemin J. Aiguier, F-13402 Marseille Cedex 20, France.
Email: [email protected] *Contributed equally to this paper and should be considered as first coauthors.
Abstract
Olfactory receptors (ORs) are the largest member of the G-protein-coupled receptors which mediate early olfactory perception in
discriminating among thousands of odorant molecules. Assigning odorous ligands to ORs is a prerequisite to gaining an understanding of the mechanisms of odorant recognition. The functional expression of ORs represents a critical step in addressing
this issue. Due to limitations in heterologous expression, very few mammal ORs have been characterized, and so far only one is
from human origin. Consequently, OR function still remains poorly understood, especially in humans, whose genome encodes
a restricted chemosensory repertoire compared with most mammal species. In this study, we have designed cassette baculovirus
vectors to coexpress human OR 17-209 or OR 17-210 with either Gaolf or Ga16 proteins in Sf9 cells. Each OR was found to be
expressed at the cell surface and colocalized with both Ga proteins. Using Ca2+ imaging, we showed that OR 17-209 and OR 17210 proteins are activated by esters and ketones respectively. Odorant-induced calcium response was increased when ORs were
coexpressed with Ga16 protein ,whereas coexpression with Gaolf abolished calcium signaling. This strategy has been found to
overcome most of the limitations encountered when expressing an OR protein and has permitted odorant screening of functional
ORs. Our approach could thus be of interest for further expression and ligand assignment of other orphan receptor proteins.
Key words: baculovirus, calcium imaging, confocal microscopy, GPCR, insect cell system, olfactory receptor
Introduction
Early olfactory discrimination of higher vertebrates is accomplished via the recognition of odorous ligands by olfactory receptors (ORs) which belong to the G-protein-coupled
receptor (GPCR) superfamily (for review, see Breer, 1994). A
single OR could bind different odorants sharing the same
odotope while one odorant molecule could be recognized
by multiple ORs, suggesting the existence of a combinatorial
receptor scheme to encode odorous entities (Malnic et al.,
1999). Odorant discrimination has been shown not only to
proceed through the recognition of numerous molecules
of widely different structures but also to markedly vary upon
odorant concentration (Kajiya et al., 2001).
Over the last decade, thousand of sequences of putative
ORs have been made available in the Olfactory Receptor
Data Bank (http://senselab.med.yale.edu/senselab/ORDB/
default.asp), which currently contains the largest family of
heptahelical receptors, covering up to 3% of the genome superfamily (for review, see Mombaerts, 2001). The human OR
genome includes numerous dysfunctional sequences, or
pseudogenes (Rouquier et al., 1998; Menashe et al., 2003),
since mutations disrupting OR coding region have been accumulated fourfold faster than any other species sampled
(Gilad et al., 2003). About 350 genes in our olfactory repertoire are potential candidates for full-length OR sequences
(Zozulya et al., 2001; Malnic et al., 2004), while almost
50% are pseudogenes (Malnic et al., 2004). Such a limited
repertoire is of particular biological relevance since the
human ortholog corresponding to an identified mammal
OR could be nonfunctional (Sharon et al., 1999; Gaillard
et al., 2002). Furthermore, not all ORs play a role in perception since human OR genes are also expressed in nonolfactory tissues, like testicular tissue, where they might be
Chemical Senses vol. 30 no. 3 ª The Author 2005. Published by Oxford University Press. All rights reserved.
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196 V. Matarazzo et al.
involved in the fertilization process (Vanti et al., 2003; Volz
et al., 2003). In this respect, we have recently shown that
the expression of two human OR genes, OR 17-209 and
OR 17-210, was restricted to the olfactory neuroepithelium
(Matarazzo et al., 2002). Both ORs are located in chromosome 17 OR gene cluster, a region of special interest as it
originates from a copy of an ancestral OR repertoire
(Ben-Arie et al., 1994). With their rather restricted chemosensory repertoire, humans represent a good model for understanding olfaction because of the intensive information
available on olfactory faculties in Homo sapiens, gathered
from earlier work (Beets, 1971) to the more recent modelling
of human olfactory stimulus–response function (Chastrette
et al., 1998), including data on phylogenic evolution of
human ORs (see http://bioportal.weizmann.ac.il/HORDE/).
Starting from gene sequences provided by genome analysis,
identifying OR proteins and assigning their ligands represent
a crucial step in the elucidation of the combinatorial olfactory code. However, functional properties of ORs still remain poorly understood since no more than a few dozen
mammal OR proteins have been successfully obtained so
far, including only one receptor of human origin. This receptor, OR 17-40, has been expressed in mammalian cells and its
ligand assigned by screening of odorant libraries (Hatt et al.,
1999; Wetzel et al., 1999). It was found to discriminate an
aldehydic molecule, helional, between structurally related
odorants.
Limitation in heterologous expression is due to impaired
OR protein translocation to the cell surface (Ivic et al.,
2002; Lu et al., 2003) and largely accounts for the limited
functional data on OR obtained so far. Among the heterologous systems available for gene expression, the Sf9 cell system has been established as a useful intact cell setting to
reconstitute GPCRs for ligand binding studies (Butkerait
et al., 1995; Barr et al., 1997). The choice of such a system
is of particular interest for human protein expression since
Sf9 cells carry out a variety of co- and post-translational processing events (Butkerait et al., 1995; Poul et al., 1995). These
processing events support viable targeting of receptors and
G-protein subunits to the cell membrane. In distinction of
mammalian cells, the Sf9 cell model is free of potential interference with endogeneous GPCRs and the low level of endogeneous G proteins permits to test the interaction of
mammalian subunits with the coexpressed receptor. Also,
the insect cells can be used for the functional expression
of mammalian receptors linked to changes in Ca2+ homeostasis (Hu et al., 1994). To provide tools for OR deorphanization, we have thus developed a strategy based on the
expression of human OR protein in Sf9 cells together with
Ga protein in order to transduce a measurable biological signal in response to odorant stimulation. We designed
recombinant baculoviruses to coexpress OR 17-209 or OR
17-210 with either Gaolf protein, a subunit involved in olfactory transduction through cAMP production (Jones and
Reed, 1989), or Ga16 protein, an ubiquitous subunit of the
phosphoinositide pathway. Quantitative pharmacological
odorant screening of both ORs has been then performed using a Ca2+ imaging method to monitor change in intracellular Ca2+ concentration in Sf9 cells.
Material and methods
Chimera constructs
The cDNA encoding rat Gaolf protein sequence was
cloned by RT-PCR using a whole-brain total RNA preparation, kindly provided by Dr. Gilles Toumaniantz (IPMC,
Nice, France). RNAs (4 lg) were primed (5 min at 70C
and 5 min at 4C) with 1 lg of oligo dT primer (Invitrogen,
Netherlands) in a final volume of 15 ll. A 10 ll aliquot of
RNA was used as a template for RT (42C for 70 min) to
cDNA using the cDNA cycle kit from Invitrogen. The reaction was then inactived at 94C for 5 min. Fisrt-strand cDNA
synthesis was used for further PCR amplification. The HA
sequence was also introduced by PCR in frame with the methionine of the Gaolf gene. The following oligonucleotic sense
(S) and antisense (AS) primers were used: Gaolf /BglII HA S: 5#TTACGATATCAGATCTGCCACCATGTACCCCTACGACGTCCCTGACTCGCCATGGGGTGTTTG-GGCAAC3#; Gaolf /HindIII AS: 5#-CTATAAGCTTTCACAAGAGTTCGTACTGCTTGAG-3#.
The cDNA encoding human Ga16 gene was obtained from
the Human Leukemia, promyelocytic HL60 cDNA library
(Clontech, France). The HA sequence was introduced by
PCR in frame with the methionine at the 5# end of cDNA.
The following oligonucleotidic sense (S) and antisense (AS)
primers were used (Eurogentec, Belgium): Ga16/BglII HA S:
5#-TTACGATATCAGATCTGCCACCAT-GTACCCCT
ACGACGTCCCT-GACTACGCCA-TGGCCCGCTCGC
TGACC-3#; Ga16/HindIII AS: 5#-CTATA-AGCTTTCACAGCA-GGTTGATCTCGTCCAG-3# (restriction sites
are underlined and HA sequence is double underlined).
The intronless genes encoding OR 17-209 and OR 17-210
were amplified by PCR using the cosmid no. ICR
F105cF06137 (GenBank HSU53583) as described previously
(Matarazzo et al., 2002). The following oligonucleotidic S
and AS primers (Eurogentec) were used: OR 17-209/NcoI
S: 5#-TAAGAAGCTTGCCACCATGGAGGGGAAAAATCTG-3#; OR-209/KpnI AS: 5#-TAACGGTACCGCGGCCGCCTAAGGGGAATGAATTTTCCG-3#; OR
17-210/NcoI S: 5#-CAATAAGCTTCCATGGCTATGTAT-TTGTGTCTCAGCAAC-3#; OR-210/KpnI AS: 5#TAACGGTACCG-CGGCCGCTTAAGCCACTGATTTAGAGTG-3#. In all PCR experiments, 10 ng of cDNA, 10
mM Tris–HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 0.001%
gelatin, 0.2 mM dNTP, 1.5 U of high-fidelity Expand mix
of polymerases (Roche Molecular Biochemicals) and 100
pmol of each primer were mixed according to the following
schedule: 94C (60 s, 1 cycle), 94C (45 s), 58C (45 s), 72C
(90 s, 35 cycles) and 72C (120 s, 1 cycle). PCR products were
Two Human ORs Expressed in a Baculovirus Insect Cell System 197
then digested and subcloned into the transfer vectors p119
and pGmAc 217T respectively.
Transfer vectors
The unique BglII and HindIII cloning sites of the p119 transfer vector (Marchal et al., 2001), located downstream of
the baculovirus p10 promoter, were used to insert either
the HA-Gaolf cDNA or HA-Ga16 cDNA. Two plasmids containing the p10 promoter, namely p119-HA-Gaolf and
p119-HA-Ga16, were thus obtained. An 108 pb oligonucleotide, corresponding to the EGT (ecdysteroid UDPS-glycosyltransferase) signal peptide (Riegel et al., 1994)
from AcMNPV followed by the FLAG epitope tagging
sequence and two unique restriction sites (NcoI and KpnI),
was inserted into the BglII/SacI sites of the pGmAc217T
transfer vector (Gaymard et al., 1996). The OR 17-209 or
OR 17-210 sequences were then inserted into the NcoI
and KpnI sites. Two plasmids containing the polyhedrin
promoter were thus obtained: pGmAc217T-FLAG-OR
17-209 and pGmAc-FLAG-OR 17-210.
was assessed by EcoRI digestion and Southern blot analysis.
Finally, in order to obtain double-recombinant baculo
viruses, AcSLP10GaolfPh or AcSLP10Ga16Ph DNA were
cotransfected with pGmAc 217T-transfer vector into Sf9 cells
and selected as obÿ virus phenotype. The final four doublerecombinant baculoviruses were designated as AcGaolf-209,
AcGa16–209, AcGaolf-210 and AcGa16–210.
Western blot analysis
Lysates (TE buffer: 10 mM Tris–HCl, pH 8.0, 1 mM EDTA)
of 36–48 h post-infected cells were electrophoresed on 12.5%
SDS–polyacrylamide gel and transferred to nitrocellulose filter papers. After a blocking step (30 min in TS buffer, 5% dry
milk, 0,05% Tween 20), blots were incubated overnight at
4C with an 1:50 dilution of anti-HA monoclonal antibody
3F10 (Roche) or 1:1000 dilution of anti-FLAG M2 monoclonal antibody (Sigma) and revealed with alkaline phosphataseconjugated goat anti-mouse IgG antibody (Promega) at 1:500
dilution. Peroxidase-conjugated antibody (Sigma) was used to
probe the in-house anti-polyhedrin antibody.
Recombinant baculoviruses
Immunofluorescence and confocal microscopy
Sf9 insect cells (ATCCCRL 1711), maintained at 28C in medium containing 5% serum, were used for virus propagation,
transfection, plaque assays and limiting dilutions. Baculoviruses DNA from AcMNPV wild type and AcSLP10 (Blanc
et al., 1993) were prepared from budding particles. Transfections were performed as previously described (Poul et al.,
1995) with minor modifications. Sf9 cells were cotransfected
with AcMNPV baculovirus and then with pGmAc217T (see
Figure 1B). This recombination procedure permitted to
obtain two single baculoviruses expressing either the
FLAG-OR 17-209 or the FLAG-OR 17-210 under control
of the polyhedrin promoter, which were plaque-purified as
an occlusion-bodies negative (obÿ) virus phenotype and designated as AcMNPV209 and AcMNPV210 respectively. A
three-step recombination procedure (see Figure 1C) was carried out to construct four double-recombinant baculoviruses
(FLAG-OR 17-209 with either HA-Gaolf or HA-Ga16 and
FLAG-OR 17-210 with either HA-Gaolf or HA-Ga16).
The first step consisted of cotransfecting Sf9 cells with
AcSLP10 baculovirus and p119 transfer vector. Recombinant baculoviruses expressing either HA-Gaolf or HA-Ga16,
under the control of the p10 promoter, were plaque-purified
as obÿ virus phenotype and designated as AcSLP10Gaolf
and AcSLP10Ga16. In a second step, AcSLP10Gaolf or
AcSLP10Ga16 DNA were cotransfected with the pGmAcI50
transfer vector (including the polyhedrin sequence and its
promoter, as illustrated in Figure 1A) into Sf9 cells. Recombinant baculoviruses expressing either Ga protein and the
polyhedrin were plaque-purified ob+ virus phenotype and
designated as AcSLP10GaolfPh and AcSLP10Ga16Ph. Insertion of polyhedrin gene in the recombinated viral genome
All fixation steps were made on ice to prevent receptor internalization. Post-infected cells (36 h) were fixed in 3% of PFA
buffer for 20 min and washed with a 0.05 M NH4Cl solution
for 10 min, then twice with PBS for 5 min. After a blocking
step (30 min with 10% inactivated goat serum buffer), cells
were incubated for 1 h with anti-FLAG (1:500 dilution) then
with FITC-coupled monoclonal anti-mouse (Sigma) at 1:128
dilution. For HA-tagged G-proteins, a 1:20 dilution of the
HA-rho conjugated rhodhamine (Roche) was used after
a permeabilization step with 0.1% of Triton X-100. For
the double-staining procedure, after anti-FLAG labelling,
cells were fixed and blocked again before permeabilization.
Coverslips were mounted on slides with Mowiol (Calbiochem), air-dried for 24 h prior to of fluorescent analysis with
a Leica TCS 4D confocal microscope with an oil-X63 lens.
OR cell surface expression
An indirect ELISA protocol was applied to quantify the expression of FLAG epitope-tagged OR constructs. Briefly, after a 36, 48 or 72 h transfection period, Sf9 cells were fixed,
blocked and incubated with anti-FLAG antibody (1:800
dilution). Plates were washed and incubated with a 1:500
dilution of alkaline phosphatase-conjugated goat antimouse IgG antibody (Promega) for 1h at room temperature.
After three washings, the p-nitrophenyl phosphate substrate
(2 mg/ml) diluted in ethanolamine (1 M, pH 9.8) was added
and further incubated in the dark for up to 1 h. Absorbance
was measured at 405 nm. Calibration was made using commercial preparation of bovine alkaline phospatase–FLAG
(Sigma).
198 V. Matarazzo et al.
A
polyhedrin promoter (pph)
5’
EGT
FLAG
Gα subunit
OR
HA
polyhedrin promoter
polyhedrin
P10 promoter (pP10)
3’
p119
pGmAc217T
pGmAcI50
C
B
pph
pph
AcSLP10 baculovirus
polyhedrin
polyhedrin
AcMNPV baculovirus
pP10
p119
pP10
P10
pph
AcSLP10Gα16 (Gαolf) baculovirus
pGmAc217T
HA
Gα subunit
pP10
pGmAcI50
pph
EGT
FLAG
OR
pph
polyhedrin
AcSLP10Gα16 (Gαolf) Ph baculovirus
AcMNPV209 (210) baculovirus
Gα subunit
pP10
P10
pP10
pGmAc217T
pph
cell infection
HA
EGT
FLAG
OR
AcGα16-209 (210) / AcGαolf-209 (210) baculoviruses
Gα subunit
HA
pP10
cell infection
OR 17-209 or OR 17-210
OR 17-209 or OR 17-210
FLAG
FLAG
Gαolf or Gα16 HA
Figure 1 Schematic representation of obtention of recombinant baculoviruses. (A) Transfer vectors used for recombination with baculovirus, obtention of
(B) monorecombinant baculovirus OR 17-209 or OR 17-210 and (C) double-recombinant baculovirus OR 17-209 or OR 17-210 with either Ga16 or Gaolf protein
subtype.
Measurement of intracellular calcium concentration
2+
Fura-2 fluorescence Ca imaging was used as previously described (Marcet et al., 2003) to measure [Ca2+]i in noninfected or 24 h post-infected cells. Briefly, Sf9 cells, cultured
on glass coverslips and loaded (1 h at room temperature)
with 4 lM fura-2-AM (Molecular Probes), were placed into
a microperfusion chamber (200 ll bath volume) and gravity
superfused with test solutions (1 ml/min). A new glass coverslip was used for each odorant mixture. Each test solution
was applied in the syringe linked to the perfusion chamber
with a manifold. Using a TILLvisION system (Germany),
[Ca2+]i was calculated from the formula proposed by
Grynkiewicz et al. (1985). Minimal and maximal experimental ratio values (Rmin and Rmax) were calculated for Sf9 cells.
Rmax and Rmin are the values of R under saturating (5 lM
A23187, a Ca2+-ionophore) and Ca2+-free conditions (4
mM EGTA combined with 5 lM A23187), respectively.
The [Ca2+]i of individual cells was thus calculated every 5 s
Two Human ORs Expressed in a Baculovirus Insect Cell System 199
and values were plotted versus time. Resting [Ca2+]i values of
individual Sf9 cells oscillated between 80 and 100 nM. For
comparison purposes, we determined the DCa2+ for each
cell, defined as the value of the [Ca2+]i increase after ligand
application minus the value of [Ca2+]i at the resting state. For
each experiment, the most responsive cells (5–10 cells out of
20–30 in the camera field of view) were selected. Data were
expressed as the mean ± SEM. Student’s t-test was used to
determine statistical significance. Differences were considered significant if P < 0.05.
Odorant screening
All odorants were tested at a concentration of 1% (w/w).
Stock solutions were stored at 4C and diluted (10ÿ6 M)
in MBS medium (Mes-buffered saline) before use. Odotope
screening was performed using successive sets of odorants.
First a mixture of 100 odorant or Henkel 100 mix, as detailed
by Wetzel et al. (1999), and a set of six odorants (camphor,
limonene, isoamyl acetate, acetophenone, 2-heptanone and
anisole), named the minimal set, were used. Next, the following sets of chemically defined groups were prepared: lactones
(pentadecanolide, whisky lactone), ketones (acetophenone,
camphor, b-ionone), esters (benzylacetate, 8-cineole, isobornylacetate, hexyl acetate, benzyl benzoate, isoamyle acetate,
ethyl butyrate, 2-phenyl ethyl acetate), phenols (thymol, eugenol, guaiacol), alcohols (menthol, citronellol, geraniol,
linalool, octanol, benzyl alcohol, cinnamyl alcohol, phenylethanol, 1-octanol) and aldehydes (citral, amyl cinnamaldehyde, cinnamaldehyde, piperonal, C7/C8/C11/C12/C13
aldehydes, octanal aldehyde, vanillin, para-anisalhedyde).
When a set was positively identified, individual odorant solutions of isoamyle acetate (from the ester set) and acetophenone (from the ketone set) were further prepared. In all
experiments, octopamine (Sigma) was used to stimulate an
endogenous GPCR (Hu et al., 1994) to control the intactness
of infected Sf9 cells. Thapsigargin (Sigma) was also used to
assess genuine intracellular calcium release response upon
odorant application.
Results
designated as AcMNPV209 or AcMNPV210 (Figure 1B).
To construct double-recombinant viruses, AcSLP10 baculovirus was recombined with transfer vectors p119, pGmAcI50
and pGmAc217T sequentially (Figure 1C). Four doublerecombinant baculoviruses, AcGaolf-209, and AcGaolf-210,
AcGa16-209 and AcGa16-210, were thereby designed.
PCR product analysis of OR and Ga protein DNAs is represented in Figure 2A. Southern blot analysis of the modified
AcSLP10 baculovirus DNA revealed that the polyhedrin
gene has been re-integrated in its proper locus, as compared
with the wild type baculovirus (Figure 2B), allowing proper
selection of recombinant viruses and subsequent OR cloning.
Western blot analysis of cell lysates showed that polyhedrin,
Ga subunits and OR recombinant proteins were found at the
expected size for each recombinated baculovirus (Figure 2C).
Expression and localization of ORs and Ga proteins
Cellular localization of OR and Ga proteins were assessed by
immunofluorescence using anti-FLAG and anti-HA mAbs
as presented in Figure 3. Confocal microscopy of Sf9 cells
infected with monorecombinant viruses AcMNPV209 or
AcMNPV210 showed a remarkably typical punctuated localization at the cell surface in absence of permeabilization
(Figure 3A, left panels), while cells expressing only Gaolf or
Ga16 subunits exhibited an intense cytoplasmic labelling after a required permeabilization step (Figure 3A, right panels). When the two labelling procedures were carried out
sequentially on infected cells with any of the four doublerecombinant viruses, a significant portion of the Ga protein
labelling was found to colocalized with OR staining (Figure
3B, right panel). No significant differences in OR staining
were noticed whether OR proteins were expressed alone
(Figure 3A, left panels) or together with a Ga subunit (Figure
3B, left panel). Using an in-house immunoassay with commercial anti-FLAG antibodies and a FLAG-protein calibrator, up to of 250 ng of recombinant tagged protein per 106
cells were estimated for each OR over a 96 h post-infection
period. Both the intensity of cell surface labelling and the estimation of OR production indicated a satisfactory level of
OR expression in the baculovirus/Sf9 cell system.
Human olfactory receptor expression
Baculovirus transfer vector pGmAc217T has been designed
for cloning PCR-amplified OR 17-209 or OR 17-210 cDNAs.
Each full-length OR coding sequence was included in a plasmid cassette containing the FLAG-tagged OR cloned downstream from an EGT signal sequence under the polyhedrin
promoter (Figure 1A, left). Both Gaolf and Ga16 sequences
were tagged with an HA sequence and cloned separately in
transfer vector p119 under the P10 promoter (Figure 1A, middle). Transfer vector pGmAcI50 (Figure 1A, right) was used
for double recombinating steps as described below. Recombination of transfer vector pGmAc217T with AcMNPV
baculovirus enabled monorecombinant OR baculoviruses
Functional characterization of OR 17-209 and OR 17-210
To assess the functional expression of OR 17-209 and OR 17210, we used fura-2 fluorescence Ca2+ imaging. Ratiometric
fluorescence (340/380) was used to calculate [Ca2+]i. For each
experiments, the resting [Ca2+]i in unstimulated cells was determined before odorant application (90 ± 10 nM, n = 100).
Pharmacological screening was based on the measurement of
an increase of [Ca2+]i in OR expressing cells upon application
of various sets of odorants. For each cell response to a given
odorant, the change in intracellular Ca2+ concentration
(D[Ca2+]i) was calculated by subtracting the resting value
of [Ca2+]i from the maximal value of [Ca2+]i induced by
200 V. Matarazzo et al.
A
09
-2
OR
MWM
17
10
-2
OR
17
MWM
16
Gα
MWM
olf
Gα
1 Kb
1 Kb
+
+
+
-
Gαolf
62
47.5
47.5
Gα
olf
Gα
OR
62
16
ol
ntr
Kda
Co
lf
αo
G
6
ol
ntr
α1
G
Co
Kda
MW
M
C
+
+
olf
+
-
OR
17210
-G
OR
α16
17210
-G
α
AcG
α16 -2
09(21
0)
Gα16
17209
OR
17209
-G
α1 6
OR
17209
-G
αolf
OR
17210
AcSL
P10G
αo
Polyhedrin
lf
AcSL
P10G
+
-
α16
AcMN
PV
B
AcG
αol f20
9(210
)
1 Kb
45 Kda
32.5
32.5
25
25
Figure 2 Analysis of recombinant materials. (A) PCR products of human OR 17-210 and OR-17-209 obtained from cosmid DNA (left). RT-PCR product analysis
of Ga16 (middle) and Gaolf (right) obtained respectively from human HL60 cDNA bank and a total cDNA brain preparation. (B) EcoRI enzymatic restriction and
Southern blot mapping of the incorporated polyedrin gene in each recombinated baculovirus and in the wild type baculovirus ACMNPV. (C) Western blot
analysis of polyhedrin protein expression (left), G-protein expression (middle) and OR expression (right). The control lane corresponds to Sf9 cells infected by wild
type baculovirus. Polyhedrin, Ga16 and Gaolf proteins were detected similarly in Sf9 cells infected with any of the four double-recombinant baculovirus (AcGa16209, AcGa16-210, AcGaolf-209, AcGaolf-210). For simplification purpose only two lanes, Ga16 or Gaolf, are represented in the left and middle Western blot
experiments.
the odorant application. In all experiments, stimulation of an
endogenous GPCR by octopamine has permitted to control
the intactness of infected Sf9 cells (Figures 4–6). In a first
step, Henkel 100 mixture (50 lM) was used to assess
large-scale screening of OR 17-209 or OR 17-210 expressed
in Sf9 cells. We observed that Henkel 100 elicited an increase
in [Ca2+]i for both ORs (data not shown). Screening was then
carried out by using selected chemical members of qualitative groups, consisting in a minimal set of six odorants
(camphor, limonene, isoamyl acetate, acetophenone, 2heptanone, anisole). Application of the minimal set evoked
a transient [Ca2+]i rise in cells expressing either OR 17-209
(D[Ca2+]i = 40 ± 5 nM, n = 4) or OR 17-210 (D[Ca2+]i =
50 ± 5 nM, n = 4), as illustrated in Figure 4A. To assess
whether odorant application induced a genuine calcium release signal, experiments were performed with thapsigarginpretreated Sf9 cells. Preincubation with thapsigargin (5 lM,
20 min) prior to odorant stimulation inhibited the calcium
response of Sf9 cells (Figure 4B). Also, when cells were incubated in free Ca2+ MBS medium, responses were still observed upon minimal set application (Figure 4B), supporting
the hypothesis that the measured [Ca2+] increase originated
from intracellular stores.
The identification of the odotope family was then pursued
by constructing from the composition of the Henkel 100 and
the minimal set six distinct chemical families of purified
Two Human ORs Expressed in a Baculovirus Insect Cell System 201
A
B
OR 209
OR
OR 210
G-protein
Gαolf
Gα16
OR+G-protein
OR 209/Gα16
OR 209/Gαolf
OR 210/Gα16
OR 210/Gαolf
Figure 3 Confocal microscopy images of Sf9 cells infected by recombinant baculoviruses. Cells were infected with either (A) monorecombinant or (B) doublerecombinant baculoviruses as indicated, prior to immunostaining procedure. Cells expressing OR 17-209 or OR 17-210 are visualized using a anti-FLAG mAb
(green fluorescence) while expressed Ga16 or Gaolf proteins are visualized with the anti-HA-rhodamine-conjugated mAb (red fluorescence). For both ORs,
202 V. Matarazzo et al.
A
200
octopamine
180
minimal set
[Ca2+]i (nM)
160
140
120
OR 17-210 expressing cell
OR 17-209 expressing cell
control cell
100
80
60
0
2
6
4
8
10
Time (min)
B
170
minimal set
160
150
[Ca2+]i (nM)
140
130
MBS buffer
120
110
100
Ca2+ free MBS buffer
90
preincubation with thapsigargin
80
0
0,5
1
1,5
2
2,5
Time (min)
Figure 4 Calcium response of Sf9 cells after minimal set stimulation. (A) A
typical recording of the Ca2+ response of a single control cell (uninfected or
wild type baculovirus infected) after minimal set application (50 lM to 1 mM)
is represented by the red line. For comparison purposes, the representative
response of a cell infected with OR 17-209 recombinant baculovirus (green
line) and one infected with OR 17-210 recombinant baculovirus (blue line)
after minimal set application (50 lM) are superimposed. In all experiments,
cell responsiveness was assessing by a second stimulation with octopamine
(50 lM). (B) Comparison of OR expressing cell responses to minimal set application (50 lM). Cells were incubated in different conditions: standard
buffer (MBS medium, green line), in the absence of extracellular Ca2+ (free
Ca2+ MBS medium, blue line) and after depletion of intracellular calcium
stores prior to odorant stimulation (20 min pre-treatment with 5 lM thapsigargin in MBS medium, red line).
odorants sharing the same functional group (ketone, ester,
lactone, alcohol, aldehyde and phenyl). Non-infected cells
did not respond to any application of each of the six mixtures
(Figure 5A). Tested sequentially, we found that esters induced an elevation of [Ca2+]i only in OR 17-209 expressing
cells (D[Ca2+]i = 45 ± 5 nM, n = 10) whereas ketones provoked a Ca2+ response (D[Ca2+]i = 55 ± 5 nM, n = 10) only
in OR 17-210 expressing cells (Figure 5B,C). Finally,
single odorants from the sets positively identified were tested.
Isoamyl acetate (from the ester set) and acetophenone (from
the ketone set) induced a calcium response in OR 17-209 or
OR 17-210 expressing cells respectively (Figure 5B,C, right).
Application of 50 lM of each odorant induced an elevation
of [Ca2+]i (D[Ca2+]i = 40 ± 5 nM, n = 4, for isoamyl acetate;
D[Ca2+]i = 50 ± 5 nM, n = 4, for acetophenone). Application
of 10 lM of odorant also induced a response in OR 17-209 or
OR 17-210 expressing cells, but with a slight calcium increase
for both isoamyl acetate (D[Ca2+]i = 7 ± 3 nM, n = 4) and
acetophenone (D[Ca2+]i = 12 ± 5 nM, n = 4).
Importantly, no response was observed when any of the
odorant (single or in mixture) was applied at concentration
ranging from micromolar to millimolar to control cells, i.e.
non-infected cells or cells infected with wild type baculovirus
(as illustrated in Figures 4A and 5A). Also, no odorant stimulation was observed when cells were infected with p119
baculovirus containing Gaolf or Ga16 protein (data not
shown). These results indicate that both ORs are functionally expressed and could be efficiently activated by application of a restricted combination of odorants or individual
odorous molecule. The calcium reponses are odorant specific
since only esters (single or set) activated OR 17-209 expressing cells while only ketones activated OR 17-210 expressing
cells. It is thus concluded that each OR specifically binds to
chemically distinct odorants. Ongoing study with structurally related molecules will further help to determine the fine
specificity of each OR protein for odorants.
As shown in Figure 6, esters (A) and ketones (B) induced
a significant increased Ca2+ responses (t-test; P < 0.05) in cells
coexpressing the Ga16 subunit with OR 17-209 (D[Ca2+]i = 56 ±
3 nM, n = 8 for esters) or OR 17-210 (D[Ca2+]i = 72 ± 5 nM,
n = 8 for ketones) respectively, compared with those observed in cells expressing OR alone (D[Ca2+]i = 46 ± 3 nM,
n = 10 for esters; D[Ca2+]i = 55 ± 5 nM, n = 10 for ketones).
The coexpression of the Ga16 subunit with OR 17-209 or OR
17-210 improved the Ca2+ response induced by esters or
ketones by 22 ± 1.5 and 31 ± 3% respectively. Similarly,
Ga16 coexpression induced also an improved calcium response of 20 ± 5% upon application of isoamyl acetate (Figure 6A) and 30 ± 2% upon application of acetophenone
(Figure 6B). Calcium response to the minimal set (Figure
6A,B) was also improved by ;20% in cells coexpressing
Ga16 with either OR. These findings therefore indicate that
the coexpression and coupling of the Ga16 subunit with OR
increase the odorant calcium response in Sf9 cells. Alternatively, in Sf9 cells coexpressing OR 17-209 or OR 17-210 together with the Gaolf subunit, no change in [Ca2+]i was
observed when any ligand (50 lM to 1 mM) was applied
to both ORs, while the octopamine (50 lM)-induced Ca2+
response was unaffected (Figure 6B,C, left). This observation suggests that both ORs may have been preferentially
coupled to the recombinant human Gaolf protein than to
immunostaining is visualized at the plasma membrane and shows typical punctuated localization at the cell surface when OR is expressed alone (A, left panels)
or coexpressed with either Ga16 or Gaolf protein (B, left panels). Similarly, either Ga subtype staining shows intense cytoplasm labelling (A, right panels and B,
middle panels). Superposition of immunostaining (yellow fluorescence) of cells infected with double-recombinant baculovirus reveals colocalization of OR and
Ga protein at the plasma membrane (B, right panels).
Two Human ORs Expressed in a Baculovirus Insect Cell System 203
A Control
p
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Figure 5 Odorant-induced calcium responses of Sf9 cells expressing OR 17-209 or OR 17-210. (A) Control cells (uninfected or wild type baculovirus infected) or
(B) cells expressing OR 17-209 or (C) cells expressing OR 17-210 were stimulated by different odorant sets (50 lM) or individual molecules (50 lM or 10 lM) as
indicated. For each Sf9 cell, the value of D[Ca2+]i was calculated. It represents the value of the maximal [Ca2+]i increase obtained upon odorant or octopamine
application (50 lM) minus the value of [Ca2+]i at the resting state. Data are means ± SEM for n independent experiments (n = 4–10; total number of cells = 30–120).
endogeneous Ga subunits of Sf9 cells, inducing a shift of
the Ca2+/IP3 signaling pathway to the cAMP pathway. Consequently no odorant-induced calcium response could be
detected in these conditions.
Taken together, the present results demonstrate that the
baculovirus/insect cell system coupled to Ca2+ imaging represents a valuable method to express functional human ORs
and investigate their odorant recognition properties. Ligand
screening was successfully performed and has permitted to
identify the respective odorant families of the human OR
17-209 and OR 17-210. Coexpression and coupling of OR
with Ga16 subunit of the same human origin may also significantly improved the odorant-induced Ca2+ response in the
Sf9 cell system.
Discussion
Difficulties in expressing OR in heterologous systems are
caused by impaired intracellular traffic to the surface of the
recombinant protein (McClintock et al., 1997; Lu et al.,
204 V. Matarazzo et al.
Figure 6 Odorant-induced calcium responses of Sf9 cells coexpressing OR together with Gaolf or Ga16 subunits. For OR 17-209 (A) or OR 17-210 (B), cells
expressing OR alone (left) and cells coexpressing OR with Ga16 subunit (middle) or Gaolf (right) are represented. The value of D[Ca2+]i of each cell was determined
upon odorant (50 lM) or octopamine application (50 lM). Data are means ± SEM for n independent experiments (n = 8–10; total number of cells = 60–100).
For ester and ketone molecules (single or set), t-tests between OR and OR-Ga protein responses were calculated (ns, not significant; *P < 0.05).
2003). These GPCRs have specific requirements for maturation and targeting to the plasma membrane, as hypothesized
by McClintock et al. (1997). Indeed, poor surface expression
of OR in heterologous cells is caused by a combination of ER
retention due to inefficient folding and ER export, aggregation and degradation (Lu et al., 2003). As a result, functional
properties of ORs have remained poorly understood, especially in the human, whose genome encodes a restricted chemosensory repertoire compared with other mammal species.
While the mechanisms responsible for inefficient translocation and surface expression of OR in heterologous cells are
now in part elucidated, it still remains a technological bottleneck in the field of OR production and deorphanization. Alternatively, performing odorant screening on OR expressed
in olfactory receptor neurons may suffer from several other
disadvantages. The presence of non-neuronal surrogate cells,
high level of background activity and lack of response reproducibility may prevent odorant identification (Reed et al.,
1998; Wetzel et al., 1999).
The insect cell system has been established as a useful intact
cell setting for GPCR expression. Co- and post-translational
processing events permit appropriate targeting of receptors
and G subunits to the cell membrane. The variety of protein
processing events performed by the Sf9 cell has provided
valuable examples of reconstituted GPCRs, in particular
for receptors of human origin (Parker et al., 1991; Butkerait
et al., 1995; Barr et al., 1997). The fact that folding of helical
secondary structure is better promoted at lower tempera-
tures (Sf9 cells are cultivated at 27C) is also of relevant importance when dealing with seven transmembrane segment
receptors. Nevertheless, since the successful expression of rodent OR5 (Raming et al., 1993), no other OR has been produced in this system so far. Mammalian cell lines have rather
been used as expression system with variable success, as
reported from our previous work (Matarazzo et al., 2002)
and in the literature. Based on these considerations, we
aimed at developing an alternate expression strategy and
used the baculovirus/Sf9 cell system to express our human
ORs. As described in this study, we have designed cassette
baculovirus vectors containing tagged OR gene fused to
an insect signal sequence. This specific leader sequence
was selected to facilitate the addressing of the protein to
the secretory pathway while the inherent properties of Sf9
cell allow appropriate folding of the recombinant product.
Confocal microscopy associated to immunodetection experiments showed that human ORs are well expressed at the
membrane of Sf9 cell together with a high level of receptor
protein. Our genetically engineered baculovirus cassette is
not restricted to human protein expression since OR sequences from any origin could also be expressed with the proper
inserts.
Ligand identification of engineered ORs requires the detection of an efficient coupling second messenger system that
produces a measurable response upon application of odorant molecules. Some inherent disadvantages in using calcium
imaging for odorant response measurement have been
Two Human ORs Expressed in a Baculovirus Insect Cell System 205
reported, e.g. lack of reversibility, saturation and low transfection rate preventing calcium quantification (Wetzel et al.,
1999). However, we did not encounter such limitations in our
Sf9 system. Specific calcium responses were measured with
cells expressing either OR, indicating that the necessary
use of small epitope tag (FLAG) at the N-terminus of the
expressed protein did not affect ligand binding or downstream signaling, as also demonstrated in the case of other
mammalian ORs (Ivic et al., 2002). Indeed, ester or ketone
application generated a rapid and quantitative [Ca2+]i increase, with fully reversible signals in OR 17-209 or OR
17-210 expressing cells, respectively. Earlier work on human
OR 17-40 indicated that odorant recognition was not affected by the choice of a given OR expression system (Wetzel
et al., 1999). Recently, however, it has been reported that
odotope affinity may differ according to the system used
(Levasseur et al., 2003). Such contradictory findings may
be explained by a low level of OR expression, thus reinforcing the need for an efficient expression system for screening
and identification of the odotope.
Since hundreds of different odorant molecules exist, we have
developed a strategy based on chemical dichotomy in order to
functionally characterize each OR. First, Henkel 100, a mixture of 100 diverse odorants from commercial mixtures
(Wetzel et al. 1999), was used to assess large-scale screening
of OR 17-209 and OR 17-210 expressed in Sf9 cells. Screening
was then carried out by using selected chemical members of
qualitative groups. These odorants, defined as the minimal
set, were originally determined on a neuro-olfactory basis
(Duchamp-Viret et al., 1999) and were used previously for
odorant stimulation of a mammal OR (Gaillard et al.,
2002). From there, identifying the odotope family was pursued
by constructing six distinct chemical families of purified odorants sharing the same functional group (ketone, ester, lactone,
alcohol, aldehyde and phenyl). Induced calcium responses
obtained with single members of a positive odorant set (isoamyl acetate or acetophenone from the ester and ketone sets
respectively) represent the first step in odotope identification
of both ORs. Current investigation is now aimed at refining
ligand identification with the use of other structurally related
odorous molecules and, if possible, defining the odorant specificity parameters for each expressed OR.
Odorant response obtained with cells expressing OR alone
or together with a Ga subtype confirmed that mammalian
GPCRs can couple to different Ga proteins, including
endogenous Ga proteins of Sf9 cells (Leopoldt et al., 1997).
Coexpression with the Ga16 subunit improves the odorantinduced calcium response (+20 to +30%), suggesting an efficient OR coupling. Such enhanced response obtained when
coexpressing a human OR together with a human Ga16 subunit might help to discriminate among related odorants.
Also, our observation underlines the putative role of the
Ga16 subtype protein in promoting intracellular calcium response to odorants (Krautwurst et al., 1998). Alternatively,
this preferential coupling may also result from a shut-off of
cellular genes as a result of baculovirus gene expression which
concomitantly reduced endogenous G-protein number and
favoured OR association with recombinant Ga16 protein.
No [Ca2+]i increase has been observed upon ligand application in Sf9 cells coexpressing either OR together with the Gaolf
protein. This observation might indicate that OR could interact with the Gaolf subunit (as suggested by confocal microscopy experiments) and thus activate the cAMP pathway but
not the IP3/Ca2+ pathway. Unfortunately, no increase in
cAMP level can be recorded because its endogenous level is
highly elevated in infected Sf9 cells and does not change upon
odorant application (Raming et al., 1993). It is therefore technically difficult to explore whether or not a shift from the IP3 to
the cAMP pathway occurred as a result of a putative coupling
of OR to Gaolf protein. Of interest, such a potential coupling
would be relevant to the physiological situation because odorant binding to ORs in olfactory neurons induced Gaolf protein
activation (Jones and Reed, 1989). Further work is needed to
determine whether ORs can efficiently couple to both transduction pathways and maintain a functional cross-talk in
our heterologous system, as previously reported for the mouse
OR-EG expressed in HEK cells (Katada et al., 2003). However, we cannot rule out that the over-expression of Gaolf
subunit may alternatively play a role of ÔscavengerÕ for endogenous Gb/c subunits. In this case, the lack of available Gb/c subunits may prevent OR signaling via an endogenous Ca2+
release-mediating Ga subunit.
In conclusion, our strategy for OR expression in the Sf9
model has been validated. Two ORs from distinct genetic
human subfamilies have been functionally expressed and their
respective odorant family identified by screening of odorous
sets based on calcium imaging. It is hoped that the enhanced
responses to odorants observed through efficient homologous
coupling of Ga16 subunit to OR will help further to discriminate quantitatively among competitive odorants. This in turn
will permit the refining of odotopes identified by deconvolution of structural odorant librairies. As our cassette strategy is
adaptable to different gene constructs, this system is convenient for expressing other ORs and assigning their respective
odorants. More generally, our approach may not be limited to
orphan OR and could be also useful for expression of various
GPCRs.
Acknowledgements
We thank J.M. Botto and J.C. Guillemot for technical assistance.
This work was supported by a grant (ANS ’odorant perception’)
from the Institut National de la Recherche Agronomique and part
of the work has been patented jointly by CNRS and INRA under
the accession number 0209377.
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Accepted December 22, 2004